Contrast agents

ABSTRACT

Contrast agents comprising microbubble-generating carbohydrate microparticles having a surfactant admixed within the microparticulate structure, with the proviso that the surfactant is not a C 10-20  fatty acid, are disclosed. Processes for preparing contrast agents also are disclosed.

[0001] This invention relates to novel contrast agents, moreparticularly to new microparticulate contrast agents of use indiagnostic imaging.

[0002] It is well known that ultrasonic imaging comprises a potentiallyvaluable diagnostic tool, for example in studies of the vascular system,particularly in cardiography, and of tissue microvasculature. A varietyof contrast agents has been proposed to enhance the acoustic images soobtained, including suspensions of solid particles, emulsified liquiddroplets, gas microbubbles and encapsulated gases or liquids. It isgenerally accepted that low density contrast agents which are easilycompressible are particularly efficient in terms of the acousticbackscatter they generate, and considerable interest has therefore beenshown in the preparation of gas-containing and gas-generating systems.

[0003] Initial studies involving free gas microbubbles generated in vivoby intracardiac injection of physiologically acceptable substances havedemonstrated the potential efficiency of such bubbles as contrast agentsin echocardiography; such techniques are severely limited in practice,however, by the short lifetime of the free bubbles. Interest hasaccordingly been shown in methods of generating and/or stabilising gasmicrobubbles for echocardiography and other ultrasonic studies, forexample using emulsifiers, oils, thickeners or sugars.

[0004] Techniques involving the use of sugars in ultrasound contrastagents are described in, for example, U.S. Pat. No. 4,681,119, U.S. Pat.No. 4,442,843 and U.S. Pat. No. 4,657,756, which disclose the use ofparticulate solids having a plurality of gas-filled voids and preferablyalso a plurality of nuclei for microbubble formation. EP-A-0123235 andEP-A-0122624 suggest ultrasound contrast agents consisting ofsurfactant-coated or surfactant-containing gas-containing microparticleswhich may include a variety of sugars. Where surfactant-containingmicroparticles are described, these are prepared simply by comminglingthe surfactant with the microparticulate materials, e.g. by trituration.

[0005] DE-A-3834705 proposes the use of suspensions containingmicroparticles of mixtures of at least one C₁₀₋₂₀ fatty acid with atleast one non-surface active substance, including sugars such ascyclodextrins, monosaccharides, disaccharides or trisaccharides, as wellas other polyols and inorganic and organic salts; in practice only theuse of galactose as the non-surface active material and only the use ofsaturated fatty acids are exemplified. The microparticulate materialsare typically prepared by coprecipitating the fatty acid and non-surfaceactive substance and comminuting the resulting product, e.g. using anair-jet mill.

[0006] One material of the type described in DE-A-3834705, SHU 508(Levovist®), is described in the following publications: Schlief, R. etal., Circulation Supplement III (1990) 82, p. 28; Schartl, M. et al.,Circulation Supplement III (1990) 82, p. 261; Fritzsch, T. et al.,Invest. Radiol. (1990) 25 (Suppl), pp. 160-161; Schlief, R. et al.,Echocardiography (1990) 7, pp. 61-64; Loughery, E. J. et al.,Echocardiography (1990) 7, pp. 279-292; and Smith, M. D. et al., JACC(1989) 13, pp. 1622-1628.

[0007] Gas-containing contrast media are also known to be effective inmagnetic resonance (MR) imaging, e.g. as susceptibility contrast agentswhich will act to reduce MR signal intensity. Oxygen-containing contrastmedia also represent potentially useful paramagnetic MR contrast agents.

[0008] Furthermore, in the field of X-ray imaging it has been observedthat gases such as carbon dioxide may be used as negative oral contrastagents.

[0009] A general disadvantage of most of the existinggas-containing/gas-generating particulate contrast agents such as thesugar-based agents discussed above is their relative lack of stabilityin vivo. This is a particular problem in applications such asechocardiography, where there is a need for improved contrast agentscombining sufficient stability and small microbubble size (typicallyless than about 10 μm, preferably less than about 7 μm) to permitpassage through the pulmonary capillary bed and so allow enhancedvisualisation of the left side of the heart, preferably for more thanone passage of circulation. There is accordingly a need for contrastagents which generate microbubble systems exhibiting good stabilitywhile still providing an effective level of contrast efficiency.

[0010] The present invention is based on our finding that contrastagents comprising microparticles of a carbohydrate having a surfactantadmixed therewith (but excluding the previously disclosed mixtures ofgalactose and saturated C₁₀₋₂₀ fatty acids) may be used to generatemicrobubble systems exhibiting enhanced contrast effect and/or stabilityrelative to previously proposed carbohydrate-based contrast agents. Inthe ultrasound field this may be demonstrated by, for example, in vitromeasurements of initial attenuation levels and the half lives of theattenuative effect; a useful indication of the combined effect of theseproperties is the integral obtained by determining the area under thecurve of a plot of attenuation against time.

[0011] The term “surfactant” as used herein means any compound havingamphiphilic properties capable of modifying surface tension.

[0012] Thus, according to one aspect of the present invention, there areprovided contrast agents comprising microbubble-generating carbohydratemicroparticles having a surfactant admixed within the microparticulatestructure, with the proviso that the surfactant is not a saturatedC₁₀₋₂₀ fatty acid when the microparticulate carbohydrate is galactose.

[0013] The microparticulate carbohydrate is preferably water soluble,and subject to the foregoing proviso may for example be selected fromhexoses such as glucose, fructose or galactose; disaccharides such assucrose, lactose or maltose; pentoses such as arabinose, xylose orribose; and polysaccharides such as α-, β- and γ- cyclodextrins,maltodextrin and glycogen; the term “carbohydrate” as used herein isalso intended to embrace sugar alcohols, e.g. alditols such as mannitolor sorbitol. Microparticles of the above carbohydrates will normallyhave gas present as an inclusion in the voids of their crystal structureand/or adhered to their surface, which gas may generate microbubbleswhen, for example, the microparticles are suspended or dissolved in aninjectable carrier liquid, for example water for injection, an aqueoussolution of one or more inorganic salts (e.g. physiological saline or aphysiological buffer solution), an aqueous solution of a monosaccharide(e.g. glucose or galactose) or disaccharide (e.g. lactose), or anaqueous solution of a physiologically tolerable monohydric or polyhydricalcohol (e.g. ethanol, propanol, isopropanol, ethylene glycol, propyleneglycol, glycerine or polyethylene glycol).

[0014] In addition to or alternatively to air, any biocompatible gas maybe employed in the contrast agents of the invention, for examplenitrogen, oxygen, hydrogen, nitrous oxide, carbon dioxide, helium,argon, sulphur hexafluoride and low molecular weight optionallyfluorinated hydrocarbons such as methane, acetylene or carbontetrafluoride. The term “gas” as used herein includes any substance inthe gaseous form at 37° C. The gas may be contained in the contrastagent in such a way that before use the product is non-contrast givingbut becomes-effective on administration, e.g. as a result of the gasforming microbubbles as a soluble carbohydrate matrix dissolves.

[0015] Additionally or alternatively the carbohydrate may incorporateone or more gas precursors, including carbonates and bicarbonates (e.g.sodium or ammonium bicarbonate) and aminomalonate esters.

[0016] Subject to the foregoing proviso a wide variety of surfactantsmay be used in the ultrasound contrast agents of the invention; it willof course be appreciated that the surfactant is required to bebiocompatible, i.e. that it should be physiologically tolerable in thequantities in which it is to be administered. The surfactant isadvantageously biodegradable in vivo or otherwise readily eliminablefrom the system.

[0017] The surfactant may, for example, be an amphiphilic lipid, e.g.selected from fatty acids and salts (e.g. alkali metal salts) thereof,steroid acids, sterols, phospholipids and glycolipids. Such lipidsinclude high molecular weight (e.g. C₁₀₋₅₀) straight chain saturated andunsaturated aliphatic acids, such as capric, palmitic, hexadecanedioic,stearic, linolenic, behenic, docosanedioic and melissic acids;aralkanoic acids, e.g. phenyl lower alkanoic acids such as2-phenylbutyric acid; salts of any of the foregoing acids; mono- anddi-glycerides, for example glyceryl esters of high molecular weight(e.g. C₁₀₋₅₀) aliphatic acids, such as glyceryl monolaurate; cholanicacids such as 5β-cholanic acid; cholesterol; sorbitan esters of fattyacids such as Span-type materials; high molecular weight (e.g. C₁₀₋₅₀)straight chain aliphatic alcohols such as stearyl alcohol and cetylalcohol; phospholipids such as phosphatidyl choline (lecithin) anddioleoylphosphatidyl ethanolamine (DOPE); and mixtures thereof

[0018] Other surfactants which may be employed include anionicsurfactants, for example alkali metal alkyl sulphates such as sodiumlauryl sulphate and sulphonated esters such as sodium dioctylsulphosuccinate (docusate); and non-ionic surfactants, for examplepolyoxyethylene-polyoxyproplyene copolymers (e.g. poloxamers such asPluronic F68) and polyoxyethylated sorbitan esters (e.g. polysorbatessuch as Tween-type materials).

[0019] The surfactant moiety may if desired be covalently linked to asubstrate such as a carbohydrate prior to its admixture with theprincipal microparticulate carbohydrate. Thus, for example, a fatty acidsuch as palmitic acid (preferably in the form of a reactive derivativesuch as a corresponding acyl halide) may be used to esterify a(preferably appropriately O-protected) sugar such as galactose and theresulting lipophilically modified carbohydrate used as the surfactant inaccordance with the invention.

[0020] The surfactant may, for example, be present in an amount of0.01-5.0 wt. %, preferably 0.1-2.0 wt. %, relative to themicroparticulate carbohydrate.

[0021] The contrast agents of the invention may be used in a variety ofdiagnostic imaging techniques, including ultrasound, MR and X-rayimaging. Their uses in diagnostic ultrasonic imaging and MR imaging,e.g. as susceptibility contrast agents, constitute preferred features ofthe invention.

[0022] The contrast agents of the invention may be prepared by anyconvenient method which leads to physical admixture of the surfactantwithin the microparticulate structure of the carbohydrate and toproduction of microparticles of the desired size.

[0023] In one preferred method according to the invention thecarbohydrate and the surfactant are each dissolved in appropriatemutually miscible solvents (e.g. water in the case of the carbohydrateand a lower alkanol such as ethanol in the case of lipid surfactantssuch as fatty acids), the resulting solutions are mixed, the solventsare removed (e.g. by evaporation under reduced pressure), and theresulting solid mixture is micronised to yield the desiredmicroparticles. It will be appreciated that all such operations shouldbe effected under sterile conditions.

[0024] In an alternative method according to the invention a (preferablyaqueous) solution of the carbohydrate is mixed with a liposome-formingmaterial (e.g. a thin film of a lipid such as lecithin formed on theinner surface of the mixing vessel by evaporating the solvent from asolution of the lipid in an appropriate organic solvent, for example achlorinated hydrocarbon such as chloroform) so as to form aliposome-containing carbohydrate solution from which the solvent may beremoved (e.g. by freeze-drying) to yield a product comprisingcarbohydrate-containing liposomes; this product may be micronised togiven microparticles of the desired size.

[0025] In general conventional micronisation techniques such as grindingor milling may be employed in processes according to the invention.Ball-milling of the solid mixture has been found to be particularlyadvantageous, permitting the preparation of microparticles in the formof aggregates (for example having an aggregate size of 20-125micrometres, such as 30-50 micrometres) of particles having a particlesize of, for example, 1-50 micrometres, such as 1-10 micrometres. Suchaggregates will tend to contain a substantial volume of air adsorbed ontheir surfaces and entrained in voids such as interparticle cavities orat grain boundaries between the crystallites. The particle size may, forexample, be selected to be substantially commensurate with the desiredmicrobubble size. In ultrasonic applications such as echocardiography,in order to permit free passage through the pulmonary system and toachieve resonance with the preferred imaging frequencies of about 0.1-15MHz, it may be convenient to employ microbubbles and microparticleshaving an average size of 0.1-10 μm, e.g. 1-7 μm; the use ofmicroparticles of average size 1-4 μm to generate microbubbles with anaverage size of 4-7 μm is generally advantageous. Substantially largerbubbles and particles, e.g. with average sizes up to 500 μm, may howeverbe useful in other applications, for example gastrointestinal imaging.

[0026] Ultrasound contrast agents in the form of microparticlescomprising a microbubble-generating carbohydrate in admixture with anamphiphilic organic acid containing in excess of 20 carbon atoms are thesubject matter of our international patent application cofiled herewithand claiming priority from British patent application No. 9200387.0.

[0027] The following non-limitative Examples serve to illustrate theinvention:-

EXAMPLES 1-18 General Procedure

[0028] D-(+)-galactose (10.0 g) was dissolved in distilled water (14.2g) at 50° C., sterile filtered and cooled on ice to a temperature of4-8° C. The stated amounts of the surfactants (in % w/w relative to thegalactose) listed in Table I were each dissolved in the amount of 96%ethanol (or water in Examples 5 and 6) shown in the Table, at 50-78° C.,and the resulting solution was sterile filtered and then asepticallyadded to the cold aqueous galactose solution under stirring. Theresulting mixture was evaporated to dryness under reduced pressure (10torr, 40° C.), and the resulting solid product was dried in a desiccatorovernight and then ground for 10 minutes under aseptic conditions in astainless steel ball mill having a 50 ml grinding cup and 3×20 mm balls(Retsch centrifugal ball mill, S1) The ground product vias dried in adesiccator for 24 hours. TABLE I Amount of Amount of ethanol ExampleSurfactant (or water) No. Surfactant (% w/w) (g) 1 Lecithin 1.0 1.2 2Lecithin 0.2 1.2 3 Sodium Lauryl Sulphate 1.0 1.0 (water) 4 SodiumLauryl Sulphate 0.1 1.0 (water) 5 Span 80 1.0 1.2 6 Span 80 0.1 1.2 7Span 85 1.0 1.2 8 Span 85 0.1 1.2 9 Pluronic F68 1.0 1.2 10 Pluronic F680.1 1.2 11 Sodium Docusate 1.0 1.2 12 Sodium Docusate 0.1 1.2 13 DOPE1.0 1.2 14 DOPE 0.1 1.2 15 α-Glyceryl Monolaurate 0.2 3.2 GlycerylTripalmitate 0.2 Cholesterol 0.2 Cholesterol Acetate 0.2 CholesterolBenzoate 0.2 16 α-Glyceryl Monolaurate 0.02 1.2 Glyceryl Tripalmitate0.02 Cholesterol 0.02 Cholesterol Acetate 0.02 Cholesterol Benzoate 0.0217 Hexadecanedioic Acid 0.2 1.2 18 Linolenic Acid 1.0 1.2

EXAMPLES 19-22

[0029] The general procedure for Examples 1-18 was repeated except thatthe D-(+)-galactose was replaced by the carbohydrates listed in TableII, in the amounts and using the quantities of water shown, and that thesurfactant used was palmitic acid (0.2% w/w relative to thecarbohydrate) dissolved in 96% ethanol (1.2 g). TABLE II Amount ofAmount of Example Microbubble-generating Carbohydrate water No.Carbohydrate (g) (g) 19 Xylose (BDH) 10.0 14.2 20 Maltodextrin 10.0 14.221 Glycogen (Merck) 5.0 17.2 22 α-Cyclodextrin (Sigma) 5.0 12.2

EXAMPLE 23 6-0-Palmitoyl-D-galactopyranose/galactose mixtures

[0030] (A) 6-O-Palmitoyl-1,2,3,4-diisopropylidene-D-galactopyranose

[0031] 1,2,3,4-Diisopropylidene-D-galactopyranose (Sigma, 13.4 g, 51.3mmol) and triethylamine (7.15 ml, 51.3 mmol) were dissolved in methylenechloride (150 ml) and cooled to 0° C. Palmitoyl chloride (Aldrich, 14.1g, 51.3 mmol) dissolved in methylene chloride (100 ml) was addeddropwise with stirring over 1 h. The cooling bath was removed and thereaction mixture was stirred overnight. Precipitated triethylaminehydrochloride was removed by filtration, the filtrate was transferred toa separating funnel and extracted with water (3×50 ml), dried over MgSO₄and the solvent was removed in vacuo. The residue was a light brownishoil which solidified to waxy crystals. Crude yield: 23 g. The crudeproduct was used without further purification. A small aliquot wasrecrystallized for characterisation. FT-IR:CO-1734 cm⁻¹. ¹³C-NMR:CO-ester 1,72.79. Mp. 124-127° C.

[0032] (B) 6-O-Palmitoyl-D-galactopyranose

[0033] 6-O-Palmitoyl-1,2,3,4-diisopropylidene-D-galactopyranose (6g) wasdissolved in acetic acid (25 ml) and heated to 100° C. under nitrogenfor 6 h. During subsequent cooling to room temperature, the productprecipitated from the solvent, and was left at room temperatureovernight. The crystals were collected by filtration and dried undervacuum. Yield: 3.3 g. The product was characterized by FT-IR: CO-1734cm⁻¹; OH-3464 cm⁻¹.

[0034] (C) 6-O-Palmitoyl-D-galactopyranose/galactose mixtures

[0035] (i) D-(+)-galactose (2 g) was dissolved in purified water (2.87g) and sterile filtered. 6-O-Palmitoyl-D-galactopyranose (0.25 g)prepared as described in (B) above was dissolved in ethanol (3 g) andsterile filtered. The solution of the palmitoyl-galactopyranose wasadded to the galactose solution under stirring and the whole mixture wastaken to dryness under vacuum (10 torr, 50° C). The product was dried ina desiccator overnight.

[0036] (ii) The procedure of (i) was repeated using6-0-palmitoyl-D-galactopyranose (0.50 g) dissolved in ethanol (6 g).

Example 24 Freeze-dried Liposomes Containing D-(+)-galactose Particles

[0037] 1 ml 100 mg/ml phosphatidylcholine was dissolved in 10 mlchloroform. The mixture was poured into a round bottom flask, and theorganic phase was evaporated at 40° C. in such a way that a thin film ofthe phosphatidylcholine was formed on the inner surface of the flask. 10ml of a sterile, pyrogen free 40% aqueous D-(+)-galactose solution wasthen added at 40° C. and the flask was kept rotating for 1 hour. Theaqueous solution containing liposomes and dissolved galactose was thenfreeze-dried for 24 hours, and the resulting product consisting offreeze-dried galactose and freeze-dried galactose-filled liposomes wasthen ground in a ball-mill to yield a product with a particle sizedistribution of 1-20 μm.

EXAMPLE 25 Echogenicity in vitro

[0038] 10 ml of propylene glycol mixed with 90 ml of 5% dextrose inwater was used as a carrier liquid for determining the echogenicity ofproducts according to the Examples. 1.0 g of each product to be testedwas dispersed in 3.0 ml of the carrier liquid and shaken for 15 seconds.The resulting mixture was added to 52 ml of 5% human serum albumininfusion solution in the measurement cell and the acoustic effects ofthe products were investigated by measuring the acoustic transmissionthrough the samples using a 5 MHz broadband transducer in apulse-reflection technique. The temperature in the measurement cell wasstabilised to 37° C. and circulation of the liquid was maintained bymeans of stirring at a constant rate. Ultrasound transmission throughthe samples was measured as a function of time over a duration of 390seconds. Results were normalized to measurements on a referenceconsisting of 55 ml of 5% human serum albumin infusion solution.

[0039] Results for representative exemplified products and comparativeresults for unmodified milled D-(+)-galactose are shown in theaccompanying drawing as FIG. 1. It will be apparent that these productsexhibit a strong effect on ultrasonic attenuation in vitro, an effectwhich persisted for several minutes.

1. A contrast agent comprising microbubble-generating carbohydratemicroparticles having a surfactant admixed within the microparticulatestructure, with the proviso that the surfactant is not a C₁₀₋₂₀ fattyacid.
 2. A contrast agent as claimed in claim 1 in which thecarbohydrate is a water-soluble pentose, hexose, disaccharide,polysaccharide or sugar alcohol.
 3. A contrast agent as claimed in claim2 in which the carbohydrate is galactose.
 4. A contrast agent as claimedin any of the preceding claims in which the surfactant is selected fromstraight chain aliphatic carboxylic acids and salts, sorbitan esters andmono- and di-glycerides thereof; aralkanoic acids and salts thereof;steroid acids; sterols; straight chain aliphatic alcohols;phospholipids; alkali metal alkyl sulphates and sulphonated esters;polyoxyethylene-polyoxypropylene copolymers; polyoxyethylated sorbitanesters and mixtures of any of the foregoing.
 5. A contrast agent asclaimed in any of the preceding claims in which the surfactant comprisesa lipophilically modified carbohydrate.
 6. A contrast agent as claimedin any of claims 1 to 4 in which the surfactant comprises carbohydrate-containing liposomes.
 7. A contrast agent as claimed in any of thepreceding claims in which the surfactant is present in an amount of0.1-2.0% w/w relative to the microparticulate carbohydrate.
 8. Acontrast agent as claimed in any of the preceding claims in which themicroparticles are aggregates having an aggregate size of 30-50micrometres of microparticles having a particle size of 1-10micrometres.
 9. A process for preparing a contrast agent as claimed inclaim 1 which comprises (i) either mixing solutions of the carbohydrateand surfactant and removing the solvent(s) therefrom or mixing asolution of the carbohydrate with a liposome-forming material andremoving the solvent therefrom and (ii) micronising the resultingmixture to yield the desired microparticles.
 10. A process as claimed inclaim 9 in which the mixture is micronised by ball-milling.
 11. Use of acontrast agent as claimed in any of claims 1 to 8 in diagnostic imaging.12. Use of a contrast agent as claimed in any of claims 1 to 8 indiagnostic ultrasonic imaging.
 13. Use of a contrast agent as claimed inany of claims 1 to 8 in magnetic resonance imaging.